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PubMed2005

Modulation of the interleukin-6 receptor subunit glycoprotein 130 complex and its signaling by LMO4 interaction.

The interleukin (IL)-6-type cytokines play major roles in a variety of biological processes by signaling through a common receptor subunit, glycoprotein (gp) 130. We performed yeast two-hybrid screening to identify new binding partners of the activated gp130 and the associated Janus kinases. LMO4, a LIM domain-containing protein that belongs to a family of oncogenes, was identified in this assay. Further studies show that LMO4 associates with gp130 and Janus kinase1 in several mammalian cell types. It also interacts with protein-tyrosine phosphatase 2 (SHP2) and suppressor of cytokine signaling 3 (SOCS3). The binding domains involved in these interactions were mapped, and the interactions were shown to be in a direct manner by in vitro binding assays. It is likely that LMO4 exists in the gp130 complex. The cellular localization of LMO4 was detected primarily in the nucleus with a substantial amount also detected in the cytoplasm in several cell types. The effect of LMO4 in IL-6 signaling was subsequently examined. Overexpression of LMO4 enhanced the transcriptional activity and target gene expression of Stat 3 (signal transducers and activators of transcription 3). Consistent with this, silencing LMO4 expression in stable cell lines expressing the small interfering RNA of LMO4 decreased Stat3 activity. Furthermore, the half-life of gp130 was shortened, and the production of acute phase proteins induced by IL-6 was reduced. Together, our data reveal a positive regulatory role of LMO4 in IL-6 signaling, possibly by acting as a scaffold for stabilization of the gp130 complex. These studies may open up a link between the oncogenic effect of LMO proteins and their regulatory role in cytokine signaling in general.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Specific decrease in the level of Hic-5, a focal adhesion protein, during immortalization of mouse embryonic fibroblasts, and its association with focal adhesion kinase.

Hic-5 is a paxillin homologue with four LIM domains in its C-terminal region, localized mainly in focal adhesions in normal fibroblasts. Hic-5 is also known to associate with focal adhesion kinase (FAK) or the related CAKbeta, and with vinculin. In the present study, we examined changes in Hic-5 and paxillin protein levels in primary mouse embryo fibroblasts (MEF) during mortal and immortal stages. The Hic-5 level was markedly decreased when cells became immortalized, whereas that of paxillin was increased. The vinculin level was not changed significantly. Hic-5 was mainly localized in focal adhesion plaques of mortal MEF but was localized in the nuclear periphery in the immortalized MEF; the number of focal adhesion plaques was decreased in these cells. Mouse Hic-5 contains three LD domains in its N-terminal half, and the first LD domain (LD1) appears to be involved in interaction with FAK. However, this interaction was not essential for recruitment of Hic-5 to focal adhesions, since its subcellular localization was similar in FAK(-/-) cells. Forced expression of Hic-5 decreased colony forming ability of MEF from FAK(+/+) mice, but not of FAK(-/-) cells. These observations suggested the involvement of Hic-5 in determination of cellular proliferative capacity in collaboration with other cytoskeletal components.

Amino Acid Sequence↗
PubMed1999

Cypher, a striated muscle-restricted PDZ and LIM domain-containing protein, binds to alpha-actinin-2 and protein kinase C.

We have cloned and characterized a novel striated muscle-restricted protein (Cypher) that has two mRNA splice variants, designated Cypher1 and Cypher2. Both proteins contain an amino-terminal PDZ domain. Cypher1, but not Cypher2, contains three carboxyl-terminal LIM domains and an amino acid repeat sequence that exhibits homology to a repeat sequence found in the largest subunit of RNA polymerase II. cypher1 and cypher2 mRNAs exhibited identical expression patterns. Both are exclusively expressed in cardiac and striated muscle in embryonic and adult stages. By biochemical assays, we have demonstrated that Cypher1 and Cypher2 bind to alpha-actinin-2 via their PDZ domains. This interaction has been further confirmed by immunohistochemical studies that demonstrated co-localization of Cypher and alpha-actinin at the Z-lines of cardiac muscle. We have also found that Cypher1 binds to protein kinase C through its LIM domains. Phosphorylation of Cypher by protein kinase C has demonstrated the functional significance of this interaction. Together, our data suggest that Cypher1 may function as an adaptor in striated muscle to couple protein kinase C-mediated signaling, via its LIM domains, to the cytoskeleton (alpha-actinin-2) through its PDZ domain.

Actinin↗
PubMed1996

Subcellular localization and protein interaction of the human LIMK2 gene expressing alternative transcripts with tissue-specific regulation.

In our efforts to explore possible roles of proteins with a LIM domain, which is a cysteine-rich Zinc-binding motif, in differentiation and oncogenesis in the lung, we have cloned a human LIMK2 gene and identified two alternative transcripts, LIMK2a and LIMK2b, which are probably due to variation in transcriptional initiation. The former encodes a protein containing two LIM domains, a PDZ domain, and a kinase domain, while the latter has only one and half LIM domains. The predominance of the two transcripts appears to be regulated in a tissue-specific manner. Alteration of the regulation is also observed in some cancer cell lines. Transfection studies have shown an association of 63-kDa and 58-kDa proteins with the LIMK2a and LIMK2b protein; the former is distributed in the cytoplasm and nucleus and the latter occurs mainly in the cytoplasm and is scarcely translocated to the nucleus. In contrast, a truncated LIMK2-Kinase has a nuclear location, not showing the protein association.

Alternative Splicing↗
PubMed2001

Intracellular signaling molecules activated by Epstein-Barr virus for induction of interferon regulatory factor 7.

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) is the principal oncogenic protein in the EBV transformation process. LMP-1 induces the expression of interferon regulatory factor 7 (IRF-7) and activates IRF-7 protein by phosphorylation and nuclear translocation. LMP-1 is an integral membrane protein with two regions in its C terminus that initiate signaling processes, the C-terminal activator regions 1 (CTAR-1) and CTAR-2. Here, genetic analysis of LMP-1 has determined that the PXQXT motif that governs the interaction between LMP-1 CTAR-1 and tumor necrosis factor receptor-associated factors (TRAFs) is needed to induce the expression of IRF-7. Mutations in the PXQXT motif in CTAR-1 that disrupt the interaction between LMP-1 and TRAFs abolished the induction of IRF-7. Also, dominant-negative mutants of TRAFs inhibited the induction of IRF-7 by CTAR-1. The last three amino acids (YYD) of CTAR-2 are also important for the induction of IRF-7. When both PXQXT and YYD were mutated (LMP-DM), the LMP-1 mutant failed to induce IRF-7. Also, LMP-DM blocked the induction of IRF-7 by wild-type LMP-1. These data strongly suggest that both CTAR-1 and CTAR-2 of LMP-1 independently induce the expression of IRF-7. In addition, NF-kappaB is involved in the induction of IRF-7. A superrepressor of IkappaB (sr-IkappaB) could block the induction of IRF-7 by LMP-1, and overexpression of NF-kappaB (p65 plus p50) could induce the expression of IRF-7. In addition, we have found that human IRF-7 is a stable protein, and sodium butyrate, a modifier of chromatin structure, induces IRF-7.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Identification and characterization of a novel protein kinase, TESK1, specifically expressed in testicular germ cells.

We have isolated cDNA clones encoding the rat and human forms of a novel protein kinase, termed TESK1 (testis-specific protein kinase 1). Sequence analysis indicates that rat TESK1 contains 628 amino acid residues, composed of an N-terminal protein kinase consensus sequence followed by a C-terminal proline-rich region. Human TESK1 contains 626 amino acids, sharing 92% amino acid identity with its rat counterpart. The protein kinase domain of TESK1 is structurally similar to those of LIMK (LIM motif-containing protein kinase)-1 and LIMK2, with 49-50% sequence identity. Phylogenetic analysis of the protein kinase domains revealed that TESK1 is most closely related to a LIMK subfamily. Chromosomal localization of human TESK1 gene was assigned to 9p13. Anti-TESK1 antibody raised against the C-terminal peptide of TESK1 recognized two polypeptides of 68 and 80 kDa in cell lysates of COS cells transfected with human TESK1 cDNA expression plasmid. TESK1 protein expressed in COS cells exhibited serine/threonine kinase activity, when myelin basic protein was used as a substrate. Northern blot analysis revealed that TESK1 mRNA was specifically expressed in rat and mouse testicular germ cells. The TESK1 mRNA in the testis was detectable only after the 18th day of postnatal development of mice and was mainly expressed in the round spermatids. These observations suggest that TESK1 has a specific function in spermatogenesis.

Alternative Splicing↗
PubMed2003

Actin binding of human LIM and SH3 protein is regulated by cGMP- and cAMP-dependent protein kinase phosphorylation on serine 146.

Various drugs that elevate cGMP levels and activate cGMP-dependent protein kinase (cGK) inhibit agonist-induced platelet activation. In the present study we identified the LIM and SH3 domain protein (LASP) that was recently cloned from human breast cancer cells (Tomasetto, C., Regnier, C., Moog-Lutz, C., Mattei, M. G., Chenard, M. P., Liderau, R., Basset, P., and Rio, M. C. (1995) Genomics 28, 367-376) as a novel substrate of cGK in human platelets. Recombinant human LASP was phosphorylated by cGMP- and cAMP-dependent protein kinase (cAK) in vitro. Cotransfection of PtK-2 cells with LASP and cGK confirmed phosphorylation of LASP in vivo. Studies with human LASP mutants identified serine 146 as a specific phosphorylation site for cGK and cAK in vivo. LASP is an actin-binding protein, and the phospho-LASP-mimicking mutant S146D showed reduced binding affinity for F-actin in cosedimentation experiments. Immunofluorescence of transfected PtK2 cells demonstrated the localization of LASP in the tips of cell membrane extensions and at cell-cell contacts. Expression of the human LASP mutant S146D resulted in nearly complete relocalization to the cytosol and reduced migration of the cells. Taken together, these data suggest that phosphorylation of LASP by cGK and cAK may be involved in cytoskeletal organization and cell motility.

Actins↗
PubMed1977

Measurement of creatine kinase Z in human sera using a DEAE-cellulose mini-column method.

A DEAE-cellulose mini-column method has been developed which allows for the quantitation in human serum of creatine kinase Z, a sub-band of creatine kinase first described by Lim ((1975) Clin. Chem. 21, 975, Abstract 181) and Sax et al. (Sax, S.M., Moore, J.J., Giegel, J.L. and Welsh, M. (1976) Clin. Chem. 22, 87). We have shown that creatine kinase Z is rather unstable in nature, and converts to a form which electrophoreses with creatine kinase MM on agarose gel electrophoresis. CK-Z is not present in normal human serum. CK-Z is present in human heart extracts, in patients with myocardial infarcts and in patients with skeletal muscle trauma. In infarct patients CK-Z levels paralleled changes in the CK-MB levels. CK-Z ranged in activity from 8.8-67.2 I.U. whereas CK-MB ranged from 29.6-121.6 I.U. in infarct patients. CK-Z and CK-MB activity were measured at or close to the peak rise in total CK activity.

Brain↗
PubMed2003

A Cypher/ZASP mutation associated with dilated cardiomyopathy alters the binding affinity to protein kinase C.

Dilated cardiomyopathy is characterized by ventricular dilation with systolic dysfunction of cardiac muscle. Recent genetic studies have revealed that mutations in genes for cytoskeleton proteins distributed in the Z-disc and/or intercalated discs of the cardiac muscle are major predictors of cardiomyopathy. However, as mutations in these genes can account for only a part of the patient population, there should be another disease-causing gene(s) for cardiomyopathy. Cypher/ZASP appears to be an ideal candidate for the cardiomyopathy causative gene, because Cypher/ZASP encodes a Z-disc associated protein, and recent studies have demonstrated that Cypher/ZASP knock-out mice develop cardiomyopathy. In this study, we searched for sequence variations in Cypher/ZASP in 96 unrelated Japanese patients with dilated cardiomyopathy. A D626N mutation located within the third LIM domain was identified in a familial case but not found in the unrelated controls. A family study of the patient showed that all affected siblings tested had the same mutation. Clinical information of the affected family members suggested that the mutation was associated with late onset cardiomyopathy. To reveal the biochemical changes due to the mutation, we performed a yeast two-hybrid assay and a pull-down assay. It was demonstrated by both assays that the D626N mutation of Cypher/ZASP increased the affinity of the LIM domain for protein kinase C, suggesting a novel biochemical mechanism of the pathogenesis of dilated cardiomyopathy.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Differential regulation of primary response gene expression in skeletal muscle cells through multiple signal transduction pathways.

One of the earliest cellular responses to growth factors is the rapid induction of primary response genes. One group of such genes was originally isolated as tetradecanoyl phorbol acetate (TPA) inducible sequences (TIS genes) from mouse 3T3 cells. Proteins encoded by the TIS genes include two transcription factors: TIS8 (also known as egr1/NGFIA/zif268) and TIS1 (also known as NGFIB/nur77/N10). We have examined the inducibility of these two genes in a skeletal muscle cell line in response to agents that have been reported to block muscle differentiation. We report here that basic fibroblast growth factor (bFGF) induced the expression of both TIS1 and TIS8 in mouse C2C12cells. Both genes were also inducible by TPA while forskolin which activates the cAMP-dependent pathway induced TIS1 but not TIS8. Down-regulation of protein kinase C (PKC) activity by TPA pretreatment repressed the bFGF induction of TIS1 but had little effect on the bFGF-stimulated expression of TIS8. Moreover, while both TPA and bFGF stimulated the hyperphosphorylation of c-RAF and the activity of MAP kinase, TPA pretreatment failed to block RAF phosphorylation or the stimulation of MAP kinase activity by bFGF. Induction of the two TIS genes in skeletal myoblasts therefore appeared to be dependent to different extents on the activation of protein kinase A (PKA), PKC and MAP kinase.

Animals↗
PubMed2004

Sho1 and Pbs2 act as coscaffolds linking components in the yeast high osmolarity MAP kinase pathway.

Scaffold proteins mediate efficient and specific signaling in several mitogen-activated protein (MAP) kinase cascades. In the yeast high osmolarity response pathway, the MAP kinase kinase Pbs2 is thought to function as a scaffold, since it binds the osmosensor Sho1, the upstream MAP kinase kinase kinase Ste11, and the downstream MAP kinase Hog1. Nonetheless, previous work has shown that Ste11 can be activated even when Pbs2 is deleted, resulting in inappropriate crosstalk to the mating pathway. We have found a region in the C terminus of Sho1 that binds Ste11 independently of Pbs2 and is required for crosstalk. These data support a model in which Sho1 has at least two separable interaction regions: one that binds Ste11 and mediates its activation, and one that binds Pbs2, directing Ste11 to act on Pbs2. Thus, a network of interactions provided by both Sho1 and Pbs2 appears to direct pathway information flow.

MAP Kinase Kinase Kinases↗
PubMedDate not supplied

Molecular cloning and characterization of a novel human kinase gene, PDIK1L.

We isolated a 4301-bp cDNA from a human foetal brain cDNA library by high-throughput cDNA sequencing. It encodes a protein of 341 amino acids, which shows 69% identity with the human kinase CLIK1 (AAL99353), which was suggested to be the CLP-36 interacting kinase. Bioinformatics analysis suggests that the putative kinase may interact with PDZ and LIM domain proteins. Therefore the protein and its cDNA were named 'PDLIM1 interacting kinase 1 like' (PDIK1L; nomenclature approved by the HUGO Gene Nomenclature Committee). Ensembl Genome Browser located PDIK1L to human chromosome 1p35.3. It spans about 13.7 kb and consists of four exons and three introns. Multiple-tissue cDNA panel PCR revealed that the gene is expressed widely in human tissues: liver, kidney, pancreas, spleen, thymus and prostate. The protein appears to be localized to the nucleus.

Amino Acid Sequence↗
PubMed1985

Phosphorylation of bovine cardiac C-protein by protein kinase C.

C-protein, a thick filament-associated protein, has been isolated from bovine myocardium and found to be a substrate in vitro of the Ca2+- and phospholipid-dependent protein kinase (protein kinase C). Incorporation of approximately 1.6 mol Pi/mol C-protein was observed. This phosphorylation was dependent on both Ca2+ and a phospholipid (L-alpha-phosphatidyl-L-serine was used). Phosphate incorporation specifically into C-protein was verified by SDS-polyacrylamide gel electrophoresis and autoradiography and was almost exclusively into serine residues (86.9%), with only a small amount of phosphothreonine (13.1%) and no phosphotyrosine being detected. Two-dimensional thin-layer electrophoresis of a chymotryptic digest of phosphorylated C-protein indicated site specificity of phosphorylation. Cardiac C-protein is known to be a substrate of cAMP-dependent protein kinase both in vitro and in vivo (Jeacocke, S.A. and England, P.J. (1980) FEBS Lett. 122, 129-132). Isolated bovine cardiac C-protein was rapidly phosphorylated, to the extent of 5 mol/mol, by the purified catalytic subunit of cAMP-dependent protein kinase. Phosphorylation catalyzed by these two protein kinases was not additive, suggesting that the sites phosphorylated by protein kinase C are also phosphorylated by cAMP-dependent protein kinase. Chicken cardiac muscle has also been shown to contain a Ca2+, calmodulin-dependent protein kinase which phosphorylates C-protein (Hartzell, H.C. and Glass, D.B. (1984) J. Biol. Chem. 259, 15587-15596). The physiological role of cardiac C-protein may therefore be subject to regulation by multiple protein kinases.

Amino Acids↗
PubMed2005

Thyrotropin-releasing hormone (TRH) specific interaction between amino terminus of P-Lim and CREB binding protein (CBP).

P-Lim (Lhx3a) is a LIM homeodomain transcription factor essential for pituitary development and motor neuron specification in mice. The Lhx3 gene encodes two isoforms, which differ in their amino (N) termini, Lhx3a and 3b. The P-Lim DNA binding site on the glycoprotein hormone alpha subunit (alpha-GSU) gene promoter is conserved in mammals. P-Lim plays a pivotal role in mediating thyrotropin-releasing hormone (TRH) signaling by binding CREB binding protein (CBP), as we have reported previously. Here, we demonstrate that P-Lim (Lhx3a) but not Lhx3b can mediate TRH signaling and bind to CBP. Moreover, TRH specifically induces P-Lim-CBP binding through the N-termini of P-Lim. We also found that the protein kinase C (PKC) phosphorylation site within the N-terminus of P-Lim is responsible for the P-Lim-CBP binding suggesting that the TRH signaling pathway phosphorylates P-Lim. These studies have elucidated the molecular mechanism by which TRH stimulates alpha-GSU gene expression.

Animals↗
PubMed2002

Roles for the tubulin- and PTP-PEST-binding paxillin LIM domains in cell adhesion and motility.

Cell dynamics mediated through cell-extracellular matrix contacts, such as adhesion and motility involve the precise regulation of large complexes of structural and signaling molecules called focal adhesions (FAs). Paxillin is a multi-domain FA adaptor protein containing five amino-terminal paxillin leucine-aspartate repeat (LD) motifs and four carboxyl-terminal Lin-11 Isl-1 and Mec-3 (LIM) domains. The LD motifs support paxillin binding to actopaxin, integrin linked kinase (ILK), FA kinase (FAK), paxillin kinase linker (PKL) and vinculin. Of the LIM domains, LIM2 and 3 comprise the paxillin FA-targeting motif, with phosphorylation of these domains modulating paxillin targeting and cell adhesion to fibronectin (Fn). The identity of the paxillin FA targeting partner remains to be determined; however, the LIM domains mediate interactions with tubulin and the protein-tyrosine phosphatase (PTP)-PEST. PTP-PEST binding requires both LIM3 and 4, whereas, the precise LIM target of tubulin binding is not known. In this report, we demonstrate that the individual paxillin LIM2 and 3 domains support specific binding to tubulin and suggest a potential role for this interaction in the regulation of paxillin sub-cellular compartmentalization. In addition, expression of paxillin molecules with mutations in the tubulin- and PTP-PEST-binding LIM domains differentially impaired Chinese hamster ovary K1 (CHO.K1) cell adhesion and migration to Fn. Perturbation of LIM3 or 4 inhibited adhesion while mutation of LIM2 or 4 decreased cell motility. Interestingly, expression of tandem LIM2-3 inhibited cell adhesion and spreading while LIM3-4 stimulated a well-spread polarized phenotype. These data offer further support for a critical role for paxillin in cell adhesion and motility.

Animals↗